Open Questions

Is a two plasmid system where the two plasmids have different antibiotic resistance genes but the same origin of replication stable? (7/16/2012)

A collaborator implied use of a different antibiotic is sufficient. However, I suppose this could lead to unpredictability in the number of each plasmid. For example, suppose the copy origin they share yields 100 plasmid copies. It could be that 10 copies of the antibiotic A & 90 copies of the antibiotic B plasmids is equally favorable as 90 copies of the antibiotic A plasmid & 10 of the antibiotic B plasmid. Presuming the two plasmids encode different proteins, the relative ratios of the expression levels would be drastically important.

Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)

Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."

online: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.

What causes an overloaded colony PCR reaction to fail?

How does plasmid copy number get affected by oxygen availability? Cellular media?

I have noticed that LB often gives me orders of magnitude lower plasmid yields and that growing cultures with liquid:air volumes higher than 1:5 is not good.

How does colony density affect colony size?

colony size varies with colony density

I observe that when there is a large number of colonies per area on an agar plate, that the colonies are smaller. I have thought about the possibility of diffusion-limited arrival of nutrients, however, one would expect to see larger colonies on the outside of the cluster if this is the case.